Burkholderia thailandensis E125 Harbors a Temperate Bacteriophage Specific for Burkholderia mallei

Abstract

Burkholderia thailandensis is a nonpathogenic gram-negative bacillus that is closely related to Burkholderia mallei and Burkholderia pseudomallei. We found that B. thailandensis E125 spontaneously produced a bacteriophage, termed E125, which formed turbid plaques in top agar containing B. mallei ATCC 23344. We examined the host range of E125 and found that it formed plaques on B. mallei but not on any other bacterial species tested, including B. thailandensis and B. pseudomallei. Examination of the bacteriophage by transmission electron microscopy revealed an isometric head and a long noncontractile tail. B. mallei NCTC 120 and B. mallei DB110795 were resistant to infection with E125 and did not produce lipopolysaccharide (LPS) O antigen due to IS407A insertions in wbiE and wbiG, respectively. wbiE was provided in trans on a broad-hostrange plasmid to B. mallei NCTC 120, and it restored LPS O-antigen production and susceptibility to E125. The 53,373-bp E125 genome contained 70 genes, an IS3 family insertion sequence (ISBt3), and an attachment site (attP) encompassing the 3 end of a proline tRNA (UGG) gene. While the overall genetic organization of the E125 genome was similar to -like bacteriophages and prophages, it also possessed a novel cluster of putative replication and lysogeny genes. The E125 genome encoded an adenine and a cytosine methyltransferase, and purified bacteriophage DNA contained both N6-methyladenine and N4-methylcytosine. The results presented here demonstrate that E125 is a new member of the supergroup of Siphoviridae that may be useful as a diagnostic tool for B. mallei.The disease glanders is caused by Burkholderia mallei, a host-adapted pathogen that does not persist in nature outside of its horse host (32). Glanders is a zoonosis, and humans whose occupations put them into close contact with infected animals can contract the disease. There have been no naturally occurring cases of glanders in North America in the last 60 years, but laboratory workers are still at risk of infection with B. mallei via cutaneous (68) and inhalational (31) routes. Human glanders has been described as a painful and loathsome disease from which few recover without antibiotic intervention (33, 51). There is little known about the virulence factors of this organism, but a recent report indicates that the capsular polysaccharide is essential for virulence in hamsters and mice (24).Burkholderia pseudomallei is the etiologic agent of the glanders-like disease melioidosis (21). As the names suggest, B. mallei and B. pseudomallei are closely related species (19,56,59,69). These ␤-Proteobacteria can now be directly compared at the genomic level because the B. pseudomallei K96243 genomic sequence is available at the Sanger Institute website (http://www.sanger.ac.uk/) and the B. mallei ATCC 23344 genomic sequence is available at the TIGR (The Institute for Genomic Research) website (http://www.tigr.org/). Preliminary BLAST (4) comparisons indicate that the genes conserved between these species are ϳ99% identical at the ...

Prophage Genomics

“…The closest match for the head genes was found in the Burkholderia siphovirus phiE125. This is a surprising finding since phiE125 was isolated from a ␤-proteobacterium (224). Over its tail genes, the most closely related phage is Shigella phage SfV.…”

Development of a polymerase chain reaction assay for the specific identification of Burkholderia mallei and differentiation from Burkholderia pseudomallei and other closely related Burkholderiaceae

“…However, Burtnick et al (2002) recently demonstrated that the lipopolysaccharide O-antigens of B. mallei and B. pseudomallei display differences in O-acetylation at position 4V of l-talose. This finding probably explains the presence of species-specific LPS O-antigen epitopes and the existence of B. mallei-specific monoclonal antibodies (Anuntagool and Sirisinha, 2002) and bacteriophages (DeShazer, 2004;Woods et al, 2002). However, these observations have not been exploited for development of rapid, simple diagnostic assays.…”

Using real-time PCR to specifically detect Burkholderia mallei

“…It should be noted that this serological test also cross-reacts with B. pseudomallei (DeShazer & Waag, 2004). To date, the only B. mallei-specific detection assay reported in the literature incorporates the bacterial phage phiE125, which has been shown to be specific for B. mallei capable of biosynthesizing LPS (Woods et al, 2002).…”